Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant inherited disease that confers an increased risk for early onset colorectal cancer. This syndrome has been linked to four recently identified DNA mismatch repair (MMR) genes. Germline mutations in each of the four MMR genes have been found in a small number of HNPCC families, but the mechanism by which these genetic aberrations cause cancers is not clear, and little is known about how specific mutations in the MMR genes correlate with the clinical manifestations of the disease. In this proposal, we hypothesize that the MMR genes are functionally tumor suppressor genes, that serve as the "afferent limb" of a DNA-monitoring system that not only recognizes and repairs DNA mismatches, but mediates G2 arrest in response to excess DNA damage. We have developed a model in which we may test the hypothesis that the MMR genes are functionally tumor suppressors. We have identified colon cancer cell lines that are deficient in mismatch repair, identified the defective gene, and have restored their ability to detect and repair DNA mismatches by the stable transfer of the relevant gene back into the cell. We propose to examine two of the MMR genes hMSH2 and hMLH1 - to determine their independent roles in mismatch repair, and to study the ability of each of these genes to recognize specific types of DNA damage. Together with collaborators, we have developed improved methods for the detection of germline mutations in patients suspected to have HNPCC, which will permit us to characterize the clinical features of tumor development based upon the genetic locus and the type of mutation involved. We also propose to determine whether the DNA mismatch repair genes function as tumor suppressor genes in vivo by looking for whether MMR deficiency provides an intrinsic growth advantage. Since the ability to recognize DNA damage and induct cell cycle arrest is mediated in part by the DNA mismatch repair genes, we propose to determine whether tumors with MMR deficiency are intrinsically resistant to the chemotherapeutic agent 5-fluorouracil. Finally, using the concept that the DNA mismatch repair gene may function like tumor suppressors, we propose a strategy to develop an in vitro screening test for HNPCC gene carriers. This application is an attempt to apply important advances in our understanding of the DNA mismatch repair genes to clinically relevant problems related to familial and sporadic colorectal cancer.